Investigations in recent years have revealed that (1.fwdarw.3)-.beta.-D-glucans have various biological activities. For instance, mention may be made of manifestation of host-mediated antitumor activity (activation of phagocytosis by macrophages, monocytes or the like, increase in bactericidal activity, etc.), activation of the mammalian alternative pathway of complement system, activation of factor G of horseshoe crab hemolymph coagulation system, and activation of the phenol oxidase system in arthropods, such as insects, and in mollusks, among others.
While the detailed mechanisms of the cell activation of (1.fwdarw.3)-.beta.-D-glucans as mentioned above are not yet clear, it is considered that specific receptors sensitive to (1.fwdarw.3)-.beta.-D-glucans exist on the cell surface and that when (1.fwdarw.3)-.beta.-D-glucans are bound to said receptors, signals are transmitted therefrom to effectors by certain cellular mechanisms to thereby cause cell activation [Goro Chihara, Yakugaku Zasshi, 108 (3), 171-186 (1988)].
The higher-order structure of a (1.fwdarw.3)-.beta.-D-glucan may vary depending on the environment in which said glucan is placed. So far known are the single helix, triple helix, random coil, and single-stranded structures. The single-stranded structure can exist only in anhydrous state. In certain instances, two or more of these higher-order structures may coexist. Carbohydrate chains with a polymerization degree of less than 20 cannot have such various higher-order structures [Saito, H. et al., Macromolecules, 11, 1244-1251 (1978)].
In the natural world, (1.fwdarw.3)-.beta.-D-glucans occur as cell wall constituents of eumycetes in considerable amount. The occurrence of proteins sensitive to (1.fwdarw.3)-.beta.-D-glucans as receptors therefor or as proenzymes in animal cells in charge of biophylaxis and required to recognize such glucans as foreign matters, for example in horseshoe crab amebocytes and immunocompetent cells such as mammalian leukocytes has been revealed and isolation of such proteins has been reported as well [Morita, T. et al., FEBS Letters, 129, 318-321 (1981); Ochiai, M. et al., J. Biol. Chem., 263, 12056-12062 (1988); Czop, J. P. et al., J. Immunol., 141, 3170-3176 (1988); and elsewhere].
Activation of cells sensitive to such (1.fwdarw.3)-.beta.-D-glucans is induced by (1.fwdarw.3)-.beta.-D-glucans or naturally occurring polysaccharides having such a structure within their molecule, or derivatives thereof. However, among these substances, some will not show such activating activity. So far, the cause of this difference has been explained in terms of the difference in degree of polymerization, branching or lack of branching within the molecule, the difference in state bound to other substances, and/or the difference in solubility in water, among others. On the other hand, a theory has been published according to which (1.fwdarw.3)-.beta.-D-glucans having a triple helix cause the activation mentioned above [Yanaki, T. et al., Agric. Biol. Chem., 50, 2415-2416 (1986)].
The present inventors observed that Sonifilan [trademark; Kaken Pharmaceutical Co., Ltd.; medicament; schizophyllan made uniform in molecular weight and higher-order structure], which has a triple helix conformation in aqueous solution, hardly activates horseshoe crab factor G and further that treatment of the above-mentioned triple helix conformation schizophyllan with an alkali to increase the single helix conformation content leads to a marked increase in the above-mentioned factor G activating activity. They further found that various (1.fwdarw.3)-.beta.-D-glucans having single helix conformation show a high activating effect on the above factor G and are also highly effective in causing regression of mouse sarcoma 180, hence have marked antitumor activity. In view of these findings, they concluded that the biological activities of (1.fwdarw.3)-.beta.-D-glucans are related to their single helix conformation [Saito, H., Yoshioka, Y., Uehara, N., Aketagawa, J., Tanaka, S. and Shibata, Y., XVth International Carbohydrate Symposium, Aug. 12-17 (1990), p. 289].
In aqueous solution, this single helix conformation of (1.fwdarw.3)-.beta.-D-glucans tends to transform into other conformations, typically a triple conformation, to bring about an equilibrium state. In particular, under high temperature and high concentration conditions, the single helix conformation is known to rapidly transform into a triple helix conformation [Kozo Ogawa, Hyomen, 12, 678-689 (1974)].
In an ordinary physiological environment, such change in higher-order structure is not so swift. For instance, when (1.fwdarw.3)-.beta.-D-glucans are observed for their biological activity in terms of horseshoe crab factor G activating ability within a short period of time, no great changes in activity are found. However, where efficacy judgment requires several days to several weeks from the first day of administration, as in antitumor activity testing in sarcoma 180, the single helix conformation of (1.fwdarw.3)-.beta.-D-glucans administered is considered to change into other higher-order structures and accordingly their biological activities decrease with time and/or the biological activities are lost during storage, leading to decreases in tumor inhibiting effect, as is often observed.